Bài giảng Molecular Biology - Chapter 15 RNA Processing II: Capping and Polyadenylation

Molecular BiologyFifth EditionChapter 15RNA Processing II: Capping and PolyadenylationLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.1RNA ProcessingEukaryotic cells perform several other kinds of processing on RNAs beyond splicingmRNAs are subject to capping at the 5’ end and polyadenylation at the 3’ end, which are essential molecular elements for the proper function of mRNA215.1 CappingBy 1974, mRNA from a variety of eukaryotic species and viruses were found to be methylatedA significant amount of this methylation was clustered at the 5’-end of mRNAThis methylation cluster formed a structure we call a cap3Cap StructureEarly study used viral mRNA as they are easier to purify and investigateThe b-phosphate of a nucleoside triphosphate remains only in the first nucleotide in an RNACap is at the 5’-terminus of RNAThe cap is made of a modified guanine or 7-methylguanosine, m7GLinkage is a triphosphate4Reovirus Cap StructureThe m7G contributes a positive chargeTriphosphate linkage contributes 3 negative chargesPhosphodiester bond contributes 1 negative chargeTerminal phosphate contributes 2 negative charges5Cap SynthesisFirst step RNA triphosphatase removes terminal phosphate from pre-mRNAThen, guanylyl transferase adds capping GMP from GTPNext, 2 methyl transferases methylate N7 of capping guanosine and 2’-O-methyl group of penultimate nucleotideThis occurs early in transcription, before chain is 30 nt long6Functions of CapsCaps serve at least four functions:Protect mRNAs from degradationEnhance translatability of mRNAsTransport of mRNAs out of nucleus Efficiency of splicing mRNAs715.2 PolyadenylationThe process of adding poly(A) to RNA is called polyadenylationA long chain of AMP residues is called a poly (A) tailHeterogeneous nuclear mRNA is a precursor to mRNA8Poly(A)Most eukaryotic mRNAs and their precursors have a chain of AMP residues about 250 nt long at their 3’-endsPoly(A) is added posttranscriptionally by an enzyme called poly(A) polymeraseTherefore, the poly (A) is not a product of transcription as it is not encoded in the DNA9Functions of Poly(A)Poly(A) enhances both the lifetime and translatability of mRNARelative importance of these two effects seems to vary from one system to anotherIn rabbit reticulocyte extracts, poly(A) seems to enhance translatability by helping to recruit mRNA to polysomes10Basic Mechanism of PolyadenylationTranscription of eukaryotic genes extends beyond the polyadenylation siteThe transcript is: CleavedPolyadenylated at 3’-end created by cleavage 11Polyadenylation SignalsAn efficient mammalian polyadenylation signal consists of:AAUAAA motif about 20 nt upstream of a polyadenylation site in a pre-mRNAFollowed 23 or 24 bp later by GU-rich motifFollowed immediately by a U-rich motifVariations on this theme occur in natureResults in variation in efficiency of polyadenylationPlant polyadenylation signals usually contain AAUAAA motifMore variation exists in plant than in animal motifYeast polyadenylation signals are even more different12Cleavage of Pre-mRNAPolyadenylation involves both:Pre-mRNA cleavagePolyadenylation at the cleavage siteCleavage in mammals requires several proteinsCPSF – cleavage and polyadenylation specificity factorCstF – cleavage stimulation factorCF ICF IIPoly (A) polymeraseRNA polymerase II13Initiation of PolyadenylationShort RNAs mimic a newly created mRNA 3’-end can be polyadenylatedOptimal signal for initiation of such polyadenylation of a cleaved substrate is AAUAAA followed by at least 8 ntWhen poly(A) reaches about 10 nt in length, further polyadenylation becomes independent of AAUAAA signal and depends on the poly(A) itself2 proteins participate in the initiation processPoly(A) polymeraseCPSF binds to the AAUAAA motif14Elongation of Poly(A)Elongation of poly(A) in mammals requires a specificity factor called poly(A)-binding protein II (PAB II)This protein Binds to a preinitiated oligo(A)Aids poly(A) polymerase in elongating poly(A) to 250 nt or morePAB II acts independently of AAUAAA motifDepends only on poly(A)Activity enhanced by CPSF15Model for PolyadenylationFactors assemble on the pre-mRNA guided by motifsCleavage occursPolymerase initiates poly(A) synthesisPAB II allows rapid extension of the oligo(A) to full-length16Poly(A) PolymeraseCloning and sequencing cDNAs encoding calf thymus poly(A) polymerase reveal a mixture of 5 cDNAs derived from alternative splicing and alternative polyadenylationStructures of the enzymes predicted from the longest sequence includes: RNA-binding domainPolymerase module2 nuclear localization signalsSer/Thr-rich region – this is dispensable for activity in vitro17Turnover of Poly(A)Poly(A) turns over in the cytoplasmRNases tear it downPoly(A) polymerase builds it back upWhen poly(A) is gone mRNA is slated for destruction18Cytoplasmic PolyadenylationCytoplasmic polyadenylation is most easily studied using Xenopus oocyte maturationMaturation-specific polyadenylation of Xenopus maternal mRNAs in the cytoplasm depends on 2 sequence motifs:AAUAAA motif near the end of mRNA Upstream motif called the cytoplasmic polyadenylation element (CPE)UUUUUAU or closely related sequence1915.3 Coordination of mRNA Processing EventsAfter reviewing capping, polyadenylation and splicing, it is clear that these processes are relatedCap can be essential for splicing, but only for splicing the first intronPoly(A) can also be essential, but only for splicing out the last intron20Processing occurs during TranscriptionAll three of the mRNA-processing events take place during transcriptionSplicing begins when transcription is still underwayCapping When nascent mRNA is about 30 nt longWhen 5’-end of RNA first emerges from polymerasePolyadenylation occurs when the still-growing mRNA is cut at the polyadenylation site21Binding of CTD of Rpb1 to mRNA-Processing ProteinsThe CTD of Rpb1 subunit of RNA polymerase II is involved in all three types of processingCapping, polyadenylating, and splicing enzymes bind directly to the CTD which serves as a platform for all three activities22CTD PhosphorylationPhosphorylation state of the CTD of Rpb1 in transcription complexes in yeast changes as transcription progressesTranscription complexes close to the promoter contain phosphorylated Ser-5Complexes farther from the promoter contain phosphorylated Ser-2Spectrum of proteins associated with the CTD also changesCapping guanylyl transferase is present early when the complex is close to promoter, not laterPolyadenylation factor Hrp1 is present in transcription complexes near and remote from promoter23RNA Processing Organized by CTD24CTD CodeIn 2007 it was demonstrated that serine 7 of the CTD can also be phosphorylatedThis raises the number of different phosphorylation states in a given repeat to eightThis raises the possibility of a ‘CTD code’ that signals for transcription of different gene sets and for different RNA modifications 25Coupling Transcription Termination with End ProcessingAn intact polyadenylation site and active factors that cleave at the polyadenylation site are required for transcription terminationActive factors that polyadenylate a cleaved pre-mRNA are not required for termination26Mechanism of TerminationTermination of transcription by RNA polymerase II occurs in 2 steps:Transcript experiences a cotranscriptional cleavage (CoTC) within termination region downstream of the polyadenylation siteThis occurs before cleavage and polyadenylation at the poly(A) siteIt is independent of that processCleavage and polyadenylation occur at the poly(A) siteSignals polymerase to dissociate from template27Termination SignalCoTC element downstream of the polyadenylation site in the human -globin mRNA is a ribozyme that cleaves itselfThis generates a free RNA 5’-endThis cleavage is required for normal transcription terminationIt provides an entry site for Xrn2, a 5’3’ exonuclease that loads onto the RNA and chases RNA polymerase by degrading the RNA28Xrn2, ExonucleaseXrn2 terminates transcription like a “torpedo”There is a similar torpedo mechanism in yeast where cleavage at poly(A) site provides entry for the 5’3’ exonuclease Rat1Rat1 degrades the RNA until it catches the polymerase and terminates transcription29Torpedo Model for Transcription Termination30Role of Polyadenylation in mRNA TransportPolyadenylation is required for efficient transport of mRNAs from their point of origin in the nucleus to the cytoplasm31